The 48 kilodalton plasminogen activator secreted by Rous sarcoma virus-infected chick embryo fibroblasts (RSV(CEF)) has been purified from conditioned media. Yields of enzyme range from 500 to 1000 micrograms per liter and are considerably higher than previously reported. Improved yield was obtained by growing the cells in medium containing small amounts of plasminogen-depleted and alpha-2-macroglobulin-depleted calf serum, as compared to the more usual approach of using serum-free medium. Purification was achieved by affinity chromatography on fibrin celite and para-aminobenzamidine agarose and by gel filtration chromatography on agarose-acrylamide beads in the presence of urea. Rabbit antibodies to the enzyme have been prepared that inhibit the plasminogen activator from (RSV (CEF)), but not from calf plasmin or human urokinase. Under our standard assay conditions, the IgG fraction of the immune serum inhibits the plasminogen activator-mediated conversion of plasminogen to plasmin at concentrations considerably less than other previously described inhibitors. Double-immunodiffusion produces a single immunoprecipitate line between the plasminogen activator and the antibodies. With adequate amounts of purified enzyme and specific antibody now available, it should be possible to determine what role, if any, plasminogen activator plays in the maintenance of the transformed state of (RSV(CEF)) and to study how synthesis of the enzyme is regulated under the control of the viral genome. (B)